General anesthetics (GAs) are known to suppress central nervous system activity in part through the activation/facilitation of postsynaptic GABA(A) receptors. While GABA(A) receptors do contribute to anesthetic action, GAs also influence presynaptic mechanisms as well. Most studies, however, have focused almost exclusively on presynaptic ion channels. Although indirect, previous experiments performed in my lab have indicated that the GAs etomidate and isoflurane inhibit the neurotransmitter release machinery directly. Amperometric data I have since collected from permeabilized PC12 cells suggests that etomidate is, in fact, capable of inhibiting the release of catecholamines via a direct interaction with the mammalian release machinery. My proposal aims to combine molecular, electrophysiological and electron microscope (EM)-based experiments to further test this hypothesis as well as identify the components of the release machinery that are involved in the response to GAs. To determine if the inhibition of the release machinery represents a universal property of GAs I will also investigate whether isoflurane affects neurotransmitter release from permeabilized PC12 cells. I will then employ photolysis of caged calcium to more accurately characterize the effects of isoflurane on the release machinery in PC12 cells. This technique allows for better control of the duration and magnitude of increases in [Ca2+]i and will allow cells to serve as their own controls. I will also determine whether syntaxin 1A or its activator, UNC-13, are involved in mediating the effects of GAs in mammalian cells. This will be done by replacing the endogenous forms of these proteins with mutants previously found to influence GA sensitivity in C. elegans. The effects of isoflurane on vesicular trafficking will also be investigated using EM. Together, these experiments have the potential to identify the release machinery as an important new target for GAs. If anesthetics were to inhibit glutamate (or other neurotransmitters) release at central synapses via this mechanism, this would provide vital information for designing new anesthetics. PUBLIC HEALTH RELEVANCE: To produce more effective general anesthetics with fewer side effects we must first understand how these anesthetics suppress activity within the mammalian nervous system. Based on my preliminary data it appears that general anesthetics are capable of suppressing neurotransmitter release in mammalian cells through a direct interaction with a group of proteins, known as SNAREs, that are responsible for synaptic vesicle fusion and neurotransmitter release. If this interaction proves to be an important part of the general action of anesthetics, this would be vital information for designing and testing new anesthetics.